The invention relates to novel compounds which bind CD11/CD18 adhesion receptors, in particular Lymphocyte Function-associated Antigen-1 (LFA-1) as well as pharmaceutical compositions containing these compounds which are useful for treating disorders mediated thereby.
Inflammation
Human peripheral blood is composed principally of red blood cells, platelets and white blood cells or leukocytes. The family of leukocytes are further classified as neutrophils, lymphocytes (mostly B- and T-cell subtypes), monocytes, eosinophils and basophils. Neutrophils, eosinophils and basophils are sometimes referred to as xe2x80x9cgranulocytesxe2x80x9d or xe2x80x9cpolymorphonuclear (PMN) granulocytesxe2x80x9d because of the appearance of granules in their cytoplasm and their multiple nuclei. Granulocytes and monocytes are often classified as xe2x80x9cphagocytesxe2x80x9d because of their ability to phagocytose or ingest micro-organisms and foreign mater referred to generally as xe2x80x9cantigensxe2x80x9d. Monocytes are so called because of their large single nucleus and these cells may in turn become macrophages. Phagocytes are important in defending the host against a variety of infections and together with lymphocytes are also involved in inflammatory disorders. The neutrophil is the most common leukocyte found in human peripheral blood followed closely by the lymphocyte. In a microliter of normal human peripheral blood, there are about 6,000 leukocytes, of which about 4,000 are neutrophils, 1500 are lymphocytes, 250 are monocytes, 150 are eosinophils and 25 are basophils.
During an inflammatory response peripheral blood leukocytes are recruited to the site of inflammation or injury by a series of specific cellular interactions (see FIG. 1). The initiation and maintenance of immune functions are regulated by intercellular adhesive interactions as well as signal transduction resulting from interactions between leukocytes and other cells. Leukocyte adhesion to vascular endothelium and migration from the circulation to sites of inflammation is a critical step in the inflammatory response (FIG. 1). T-cell lymphocyte immune recognition requires the interaction of the T-cell receptor with antigen (in combination with the major histocompatibility complex) as well as adhesion receptors, which promote attachment of T-cells to antigen-presenting cells and transduce signals for T-cell activation. The lymphocyte function associated antigen-1 (LFA-1) has been identified as the major integrin that mediates lymphocyte adhesion and activation leading to a normal immune response, as well as several pathological states (Springer, T. A., Nature 346:425-434 (1990)). Intercellular adhesion molecules (ICAM)-1, -2, and -3, members of the immunoglobulin superfamily, are ligands for LFA-1 found on endothelium, leukocytes and other cell types. The binding of LFA-1 to ICAMs mediate a range of lymphocyte functions including lymphokine production of helper T-cells in response to antigen presenting cells, T-lymphocyte mediated target cells lysis, natural killing of tumor cells, and immunoglobulin production through T-cell-B-cell interactions. Thus, many facets of lymphocyte function involve the interaction of the LFA-1 integrin and its ICAM ligands. These LFA-1:ICAM mediated interactions have been directly implicated in numerous inflammatory disease states including; graft rejection, dermatitis, psoriasis, asthma and rheumatoid arthritis.
While LFA-1 (CD11a/CD18) on lymphocytes plays a key role in chronic inflammation and immune responses, other members of the leukocyte integrin family (CD11b/CD18, CD11c/CD18 and CD11d/CD18) also play important roles on other leukocytes, such as granulocytes and monocytes, particularly in early response to infective agents and in acute inflammatory response.
The primary function of polymorphonuclear leukocytes, derived from the neutrophil, eosinophil and basophil lineage, is to sense inflammatory stimuli and to emigrate across the endothelial barrier and carry out scavenger function as a first line of host defense. The integrin Mac-1(CD11b/CD18) is rapidly upregulated on these cells upon activation and binding to its multiple ligands which results in the release of oxygen derived free radicals, protease""s and phospholipases. In certain chronic inflammatory states this recruitment is improperly regulated resulting in significant cellular and tissue injury. (Harlan, J. M., Acta Med Scand Suppl., 715:123 (1987); Weiss, S., New England J. of Med., 320:365 (1989)). LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) The (CD11/CD18) family of adhesion receptor molecules comprises four highly related cell surface glycoproteins; LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150.95 (CD11c/CD18) and (CD11d/CD18). LFA-1 is present on the surface of all mature leukocytes except a subset of macrophages and is considered the major lymphoid integrin. The expression of Mac-1, p150.95 and CD11d/CD18 is predominantly confined to cells of the myeloid lineage (which include neutrophils, monocytes, macrophage and mast cells). Functional studies have suggested that LFA-1 interacts with several ligands, including ICAM-1 (Rothleinet al., J. Immunol. 137:1270-1274 (1986), ICAM-2, (Staunton et al., Nature 339:361-364 (1989)), ICAM-3 (Fawcett et al., Nature 360:481-484 (1992); Vezeux et al., Nature 360:485-488, (1992); de Fougerolles and Springer, J. Exp. Med. 175:185-190 (1990)) and Telencephalin (Tian et al., J. Immunol. 158:928-936 (1997)).
The CD11/CD18 family is related structurally and genetically to the larger integrin family of receptors that modulate cell adhesive interactions, which include; embryogenesis, adhesion to extracellular substrates, and cell differentiation (Hynes, R. O., Cell 48:549-554 (1987); Kishimotoet al., Adv. Immunol. 46:149-182 (1989); Kishimotoet al., Cell 48:681-690 (1987); Ruoslahtiet al., Science 238:491-497 (1987).
Integrins are a class of membrane-spanning heterodimers comprising an xcex1 subunit in noncovalent association with a xcex2 subunit. The xcex2 subunits are generally capable of association with more than one xcex1 subunit and the heterodimers sharing a common xcex2 subunit have been classified as subfamilies within the integrin population (Larson and Springer, xe2x80x9cStructure and function of leukocyte integrins,xe2x80x9d Immunol. Rev 114:181-217 (1990)).
The integrin molecules of the CD11/CD18 family, and their cellular ligands, have been found to mediate a variety of cellxe2x80x94cell interactions, especially in inflammation. These proteins have been demonstrated to be critical for adhesive functions in the immune system (Kishimotoet al., Adv. Immunol. 46:149-182 (1989)). Monoclonal antibodies to LFA-1 have been shown to block leukocyte adhesion to endothelial cells (Dustin et al., J. Cell. Biol. 107:321-331 (1988); Smith et al., J. Clin. Invest. 83:2008-2017 (1989)) and to inhibit T-cell activation (Kuypers et al., Res. Immnunol., 140:461 (1989)), conjugate formation required for antigen-specific CTL killing (Kishimotoet al., Adv. Immunol. 46:149-182 (1989)), T. cell proliferation (Davignonet al., J. Immunol. 127:590-595 (1981)) and NK cell killing (Krenskyet al., J. Immunol. 131:611-616 (1983)). ICAMs
ICAM-1 (CD54) is a cell surface adhesion receptor that is a member of the immunoglobulin protein super-family (Rothleinet al., J. Immunol. 137:1270-1274 (1986); Stauntonet al., Cell 52:925-933 (1988). Members of this superfamily are characterized by the presence of one or more Ig homology regions, each consisting of a disulfide-bridged loop that has a number of anti-parallel xcex2-pleated strands arranged in two sheets. Three types of homology regions have been identified, each with a typical length and having a consensus sequence of amino acid residues located between the cysteines of the disulfide bond (Williams, A. F. et al. Ann Rev. Immunol. 6:381-405 (1988); Hunkapillar, T. et al. Adv. Immunol. 44:1-63 (1989). ICAM-1 is expressed on a variety of hematopoietic and non-hematopoietic cells and is upregulated at sites of inflammation by a variety of inflammatory mediators (Dustin et al., J. Immunol., 137:256-254 (1986)). ICAM-1 is a 90,000-110,000 Mr glycoprotein with a low messenger RNA levels and moderate surface expression on unstimulated endothelial cells. LPS, IL-1 and TNF strongly upregulate ICAM-1 mRNA and surface expression with peak expression at approximately 18-24 hours (Dustinet al., J. Cell. Biol. 107:321-331 (1988); Stauntonet al., Cell 52:925-933 (1988)). ICAM-1 has five extracellular Ig like domains (designated Domains 1, 2, 3, 4 and 5 or D1, D2, D3, D4 and D5) and an intracellular or cytoplasmic domain. The structures and sequence of the domains is described by Staunton et al. (Cell 52:925-933 (1988)).
ICAM-1 was defined originally as a counter-receptor for LFA-1 (Springer et al., Ann. Rev. Immunol, 5:223-252 (1987); MarlinCell 51:813-819 (1987); Simmonset al., Nature 331:624-627 (1988); Staunton Nature 339:61-64 (1989); Stauntonet al., Cell 52:925-933 (1988)). The LFA-1/ICAM-1 interaction is known to be at least partially responsible for lymphocyte adhesion (Dustinet al., J. Cell. Biol. 107:321-331 (1988); Mentzeret al., J. Cell. Physiol. 126:285-290 (1986)), monocyte adhesion (Amaout et al., J. Cell Physiol. 137:305 (1988); Mentzeret al., J. Cell. Physiol. 130:410-415 (1987); te Veldeet al., Immunology 61:261-267 (1987)), and neutrophil adhesion (Loet al., J. Immunol. 143(10):3325-3329 (1989); Smith et al., J. Clin. Invest. 83:2008-2017 (1989)) to endothelial cells. Through the development of function blocking monoclonal antibodies to ICAM-1 additional ligands for LFA-1 were identified, ICAM-2 and ICAM-3 (Simmons, Cancer Surveys 24, Cell Adhesion and Cancer, 1995) that mediate the adhesion of lymphocytes to other leukocytes as well as non-hematopoietic cells. Interactions of LFA-1 with ICAM-2 are thought to mediate natural killer cell activity (Helander et al., Nature 382:265-267 (1996)) and ICAM-3 binding is thought to play a role in lymphocyte activation and the initiation of the immune response (Simmons, ibid). The precise role of these ligands in normal and aberrant immune responses remains to be defined.
Disorders Mediated by T Lymphocytes
Function blocking monoclonal antibodies have shown that LFA-1 is important in T-lymphocyte-mediated killing, T-helper lymphocyte responses, natural killing, and antibody-dependent killing (Springer et al., Ann. Rev. Immunol 5:223-252 (1987)). Adhesion to the target cell as well as activation and signaling are steps that are blocked by antibodies against LFA-1.
Many disorders and diseases are mediated through T lymphocytes and treatment of these diseases have been addressed through many routes. Rheumatoid arthritis (RA) is one such disorder. Current therapy for RA includes bed rest, application of heat, and drugs. Salicylate is the currently preferred treatment drug, particularly as other alternatives such as immunosuppressive agents and adrenocorticosteroids can cause greater morbidity than the underlying disease itself. Nonsteroidal anti-inflammatory drugs are available, and many of them have effective analgesic, anti-pyretic and anti-inflammatory activity in RA patients. These include cyclosporin, indomethacin, phenylbutazone, phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, and diflunisal. Other drugs for use in RA include anti-malarials such as chloroquine, gold salts and penicillamine. These alternatives frequently produce severe side effects, including retinal lesions and kidney and bone marrow toxicity. Immunosuppressive agents such as methotrexate have been used only in the treatment of severe and unremitting RA because of their toxicity. Corticosteroids also are responsible for undesirable side effects (e.g., cataracts, osteoporosis, and Cushing""s disease syndrome) and are not well tolerated in many RA patients.
Another disorder mediated by T lymphocytes is host rejection of grafts after transplantation. Attempts to prolong the survival of transplanted allografts and xenografts, or to prevent host versus graft rejection, both in experimental models and in medical practice, have centered mainly on the suppression of the immune apparatus of the host/recipient. This treatment has as its aim preventive immunosuppression and/or treatment of graft rejection. Examples of agents used for preventive immunosuppression include cytotoxic drugs, anti-metabolites, corticosteroids, and anti-lymphocytic serum. Nonspecific immunosuppressive agents found particularly effective in preventive immunosuppression (azathioprine, bromocryptine, methylprednisolone, prednisone, and most recently, cyclosporin A) have significantly improved the clinical success of transplantation. The nephrotoxicity of cyclosporin A after renal transplantation has been reduced by co-administration of steroids such as prednisolone, or prednisolone in conjunction with azathioprine. In addition, kidneys have been grafted successfully using anti-lymphocyte globulin followed by cyclosporin A. Another protocol being evaluated is total lymphoid irradiation of the recipient prior to transplantation followed by minimal immunosuppression after transplantation.
Treatment of rejection has involved use of steroids, 2-amino-6-aryl-5-substituted pyrimidines, heterologous anti-lymphocyte globulin, and monoclonal antibodies to various leukocyte populations, including OKT-3. See generally J. Pediatrics, 111: 1004-1007 (1987), and specifically U.S. Pat. No. 4,665,077.
The principal complication of immunosuppressive drugs is infections. Additionally, systemic immunosuppression is accompanied by undesirable toxic effects (e.g., nephrotoxicity when cyclosporin A is used after renal transplantation) and reduction in the level of the hemopoietic stem cells. Immunosuppressive drugs may also lead to obesity, poor wound healing, steroid hyperglycemia, steroid psychosis, leukopenia, gastrointestinal bleeding, lymphoma, and hypertension.
In view of these complications, transplantation immunologists have sought methods for suppressing immune responsiveness in an antigen-specific manner (so that only the response to the donor alloantigen would be lost). In addition, physicians specializing in autoimmune disease strive for methods to suppress autoimmune responsiveness so that only the response to the self-antigen is lost. Such specific immunosuppression generally has been achieved by modifying either the antigenicity of the tissue to be grafted or the specific cells capable of mediating rejection. In certain instances, whether immunity or tolerance will be induced depends on the manner in which the antigen is presented to the immune system.
Pretreating the allograft tissues by growth in tissue culture before transplantation has been found in two murine model systems to lead to permanent acceptance across MHC barriers. Lafferty et al., Transplantation, 22:138-149 (1976); Bowen et al., Lancet, 2:585-586 (1979). It has been hypothesized that such treatment results in the depletion of passenger lymphoid cells and thus the absence of a stimulator cell population necessary for tissue immunogenicity. Lafferty et al., Annu. Rev. Immunol., 1:143 (1983). See also Lafferty et al., Science, 188:259-261 (1975) (thyroid held in organ culture), and Gores et al., J. Immunol., 137:1482-1485 (1986) and Faustman et al., Proc. Natl. Acad. Sci. U.S.A., 78: 5156-5159 (1981) (islet cells treated with murine anti-Ia antisera and complement before transplantation). Also, thyroids taken from donor animals pretreated with lymphocytotoxic drugs and gamma radiation and cultured for ten days in vitro were not rejected by any normal allogeneic recipient (Gose and Bach, J. Exp. Med., 149:1254-1259 (1979)). All of these techniques involve depletion or removal of donor lymphocyte cells.
In some models such as vascular and kidney grafts, there exists a correlation between Class II matching and prolonged allograft survival, a correlation not present in skin grafts (Pescovitz et al., J. Exp. Med., 160:1495-1508 (1984); Conti et al., Transplant. Proc., 19: 652-654 (1987)). Therefore, donor-recipient HLA matching has been utilized. Additionally, blood transfusions prior to transplantation have been found to be effective (Opelz et al., Transplant. Proc., 4: 253 (1973); Persijn et al., Transplant. Proc., 23:396 (1979)). The combination of blood transfusion before transplantation, donor-recipient HLA matching, and immunosuppression therapy (cyclosporin A) after transplantation was found to improve significantly the rate of graft survival, and the effects were found to be additive (Opelz et al., Transplant. Proc., 17:2179 (1985)).
The transplantation response may also be modified by antibodies directed at immune receptors for MHC antigens (Bluestone et al., Immunol. Rev 90:5-27 (1986)). Further, graft survival can be prolonged in the presence of antigraft antibodies, which lead to a host reaction that in turn produces specific immunosuppression (Lancaster et al., Nature, 315: 336-337 (1985)). The immune response of the host to MHC antigens may be modified specifically by using bone marrow transplantation as a preparative procedure for organ grafting. Thus, anti-T-cell monoclonal antibodies are used to deplete mature T-cells from the donor marrow inoculum to allow bone marrow transplantation without incurring graft-versus-host disease (Mueller-Ruchholtz et al., Transplant Proc., 8:537-541 (1976)). In addition, elements of the host""s lymphoid cells that remain for bone marrow transplantation solve the problem of immunoincompetence occurring when fully allogeneic transplants are used.
As shown in FIG. 1, lymphocyte adherence to endothelium is a key event in the process of inflammation. There are at least three known pathways of lymphocyte adherence to endothelium, depending on the activation state of the T-cell and the endothelial cell. T-cell immune recognition requires the contribution of the T-cell receptor as well as adhesion receptors, which promote attachment ofxe2x80x94cells to antigen-presenting cells and transduce regulatory signals for T-cell activation. The lymphocyte function associated (LFA) antigen-1 (LFA-1, CD11a/CD18, xe2x96xa1 xcex1Lxcex22: where xcex1L is CD11a and xcex22 is CD18) has been identified as the major integrin receptor on lymphocytes involved in these cell adherence interactions leading to several pathological states. ICAM-1, the endothelial cell immunoglobulin-like adhesion molecule, is a known ligand for LFA-1 and is implicated directly in graft rejection, psoriasis, and arthritis.
LFA-1 is required for a range of leukocyte functions, including lymphokine production of helper T-cells in response to antigen-presenting cells, killer T-cell-mediated target cell lysis, and immunoglobulin production through T-cell/B-cell interactions. Activation of antigen receptors on T-cells and B-cells allows LFA-1 to bind its ligand with higher affinity.
Monoclonal antibodies (MAbs) directed against LFA-1 led to the initial identification and investigation of the function of LFA-1 (Davignon et al., J. Immunol., 127:590 (1981)). LFA-1 is present only on leukocytes (Krenskey et al., J. Immunol., 131:611 (1983)), and ICAM-1 is distributed on activated leukocytes, dermal fibroblasts, and endothelium (Dustin et al., J. Immunol. 137:245 (1986)).
Previous studies have investigated the effects of anti-CD11a MAbs on many T-cell-dependent immune functions in vitro and a limited number of immune responses in vivo. In vitro, anti-CD11a MAbs inhibit T-cell activation (Kuypers et al., Res. Immunol., 140:461 (1989)), T-cell-dependent B-cell proliferation and differentiation (Davignon et al., supra; Fischer et al., J. Immunol., 136:3198 (1986)), target cell lysis by cytotoxic T-lymphocytes (Krensky et al., supra), formation of immune conjugates (Sanders et al., J. Immunol., 137:2395 (1986); Mentzer et al., J. Immunol., 135:9 (1985)), and the adhesion of T-cells to vascular endothelium (Lo et al., J. Immunol., 143:3325 (1989)). Also, the antibody 5C6 directed against CD11b/CD18 was found to prevent intra-islet infiltration by both macrophages and T cells and to inhibit development of insulin-dependent diabetes mellitis in mice (Hutchings et al., Nature, 348: 639 (1990))
The observation that LFA-1:ICAM-1 interaction is necessary to optimize T-cell function in vitro, and that anti-CD11a MAbs induce tolerance to protein antigens (Benjamin et al., Eur. J. Immunol., 18:1079 (1988)) and prolongs tumor graft survival in mice (Heagy et al., Transplantation, 37: 520-523 (1984)) was the basis for testing the MAbs to these molecules for prevention of graft rejection in humans.
Experiments have also been carried out in primates. For example, based on experiments in monkeys it has been suggested that a MAb directed against ICAM-1 can prevent or even reverse kidney graft rejection (Cosimi et al., xe2x80x9cImmunosuppression of Cynomolgus Recipients of Renal Allografts by R6.5, a Monoclonal Antibody to Intercellular Adhesion Molecule-1,xe2x80x9d in Springer et al. (eds.), Leukocyte Adhesion Molecules New York: Springer, (1988), p. 274; Cosimi et al., J. Immunology, 144:4604-4612 (1990)). Furthermore, the in vivo administration of anti-CD11a MAb to cynomolgus monkeys prolonged skin allograft survival (Berlin et al., Transplantation, 53: 840-849 (1992)).
The first successful use of a rat anti-murine CD11a antibody (25-3; IgG1) in children with inherited disease to prevent the rejection of bone-marrow-mismatched haploidentical grafts was reported by Fischer et al., Lancet, 2: 1058 (1986). Minimal side effects were observed. See also Fischer et al., Blood, 77: 249 (1991); van Dijken et al., Transplantation, 49:882 (1990); and Perez et al., Bone Marrow Transplantation, 4:379 (1989). Furthermore, the antibody 25-3 was effective in controlling steroid-resistant acute graft-versus-host disease in humans (Stoppa et al., Transplant. Int., 4:3-7 (1991)).
However, these results were not reproducible in leukemic adult grafting with this MAb (Maraninchi et al., Bone Marrow Transplant, 4:147-150 (1989)), or with an anti-CD18 MAb, directed against the invariant chain of LFA-1, in another pilot study (Baume et al., Transplantation, 47: 472 (1989)). Furthermore, a rat anti-murine CD11a MAb, 25-3, was unable to control the course of acute rejection in human kidney transplantation (LeMauff et al., Transplantation, 52: 291 (1991)).
A review of the use of monoclonal antibodies in human transplantation is provided by Dantal and Soulillou, Current Opinion in Immunology, 3:740-747 (1991). An earlier report showed that brief treatment with either anti-LFA-1 or anti-ICAM-1 MAbs minimally prolonged the survival of primarily vascularized heterotopic heart allografts in mice (Isobe et al., Science, 255:1125 (1992)). However, combined treatment with both MAbs was required to achieve long-term graft survival in this model.
Independently, it was shown that treatm7ent with anti-LFA-1 MAb alone potently and effectively prolongs the survival of heterotopic (ear-pinnae) nonprimarily vascularized mouse heart grafts using a maximum dose of 4 mg/kg/day and treatment once a week after a daily dose (Nakakura et al., J. Heart Lung Transplant., 11:223 (1992)). Nonprimarily vascularized heart allografts are more immunogenic and more resistant to prolongation of survival by MAbs than primarily vascularized heart allografts (Warren et al., Transplant. Proc., 5:717 (1973); Trager et al., Transplantation, 47:587 (1989)). The latter reference discusses treatment with L3T4 antibodies using a high initial dose and a lower subsequent dose.
Another study on treating a sclerosis-type disease in rodents using similar antibodies to those used by Nakakura et al., supra, is reported by Yednock et al., Nature, 356:63-66 (1992). Additional disclosures on the use of anti-LFA-1 antibodies and ICAM-1, ICAM-2, and ICAM-3 and their antibodies to treat LFA-1-mediated disorders include WO 91/18011 published Nov. 28, 1991, WO 91/16928 published Nov. 14, 1991, WO 91/16927 published Nov. 14, 1991, Can. Pat. Appln. 2,008,368 published Jun. 13, 1991, WO 90/03400, WO 90/15076 published Dec. 13, 1990, WO 90/10652 published Sep. 20, 1990, EP 387,668 published Sep. 19, 1990, WO 90/08187 published Jul. 26, 1990, WO 90/13281, WO 90/13316, WO 90/13281, WO 93/06864, WO 93/21953, WO 93/13210, WO 94/11400, EP 379,904 published Aug. 1, 1990, EP 346,078 published Dec. 13, 1989, U.S. Pat. Nos. 5,002,869, 5,071,964, 5,209,928, 5,223,396, 5,235,049, 5,284,931, 5,288,854, 5,354,659, Australian Pat. Appln. 15518/88 published Nov. 10, 1988, EP 289,949 published Nov. 9, 1988, and EP 303,692 published Feb. 22, 1989, EP 365,837, EP 314,863, EP 319,815, EP 468, 257, EP 362,526, EP 362, 531, EP 438,310.
Other disclosures on the use of LFA-1 and ICAM peptide fragments and antagonists include; U.S. Pat. Nos. 5,149,780, 5,288,854, 5,340,800, 5,424,399, 5,470,953, WO 90/03400, WO 90/13316, WO 90/10652, WO 91/19511, WO 92/03473, WO 94/11400, WO 95/28170, JP 4193895, EP 314,863, EP 362,526 and EP 362,531.
The above methods successfully utilizing anti-LFA-1 or anti-ICAM-1 antibodies, LFA-1 or ICAM-1 peptides, fragments or peptide antagonists represent an improvement over traditional immunosuppressive drug therapy. These studies demonstrate that LFA-1 and ICAM-1 are appropriate targets for antagonism. There is a need in the art to better treat disorders that are mediated by LFA-1 including autoimmune diseases, graft vs. host or host vs. graft rejection, and T-cell inflammatory responses, so as to minimize side effects and sustain specific tolerance to self- or xenoantigens.
There is also a need in the art to provide a non-peptide antagonists to the LFA-1: ICAM-1 interaction.
Albumin is an abundant plasma protein which is responsible for the transport of fatty acids. However, albumin also binds and perturbs the pharmacokinetics of a wide range of drug compounds. Accordingly, a significant factor in the pharmacological profile of any drug is its binding characteristics with respect to serum plasma proteins such as albumin. A drug compound may have such great affinity for plasma proteins that it is not be available in serum to interact with its target tissue, cell or protein. For example, a compound for which 99% binds to plasma protein upon administration will have half the concentration available in plasma to interact with its target than a compound which binds only 98%. Accordingly it would be desirable to provide LFA antagonist compounds which have low serum plasma protein binding affinity.
In an aspect of the present invention, there is provided novel compounds of formula (I) 
wherein
Cy is a non-aromatic carbocycle or heterocycle optionally substituted with hydroxyl, mercapto, thioalkyl, halogen, oxo, thio, amino, aminoalkyl, amidine, guanidine, nitro, alkyl, alkoxy or acyl;
X is a divalent hydrocarbon chain optionally substituted with hydroxyl, mercapto, halogen, amino, aminoalkyl, nitro, oxo or thio and optionally interrupted with N, O, S, SO or SO2;
Y is a carbocycle or heterocycle optionally substituted with hydroxyl, mercapto, halogen, oxo, thio, a hydrocarbon, a halo-substituted hydrocarbon, amino, amidine, guanidine, cyano, nitro, alkoxy or acyl;
L is a bond or a divalent hydrocarbon optionally having one or more carbon atoms replaced with N, O, S, SO or SO2 and optionally being substituted with hydroxyl, halogen oxo or thio; or three carbon atoms of the hydrocarbon are replaced with an amino acid residue;
R1 is H, OH, amino, O-carbocycle or alkoxy optionally substituted with amino, a carbocycle or a heterocycle;
R2-5 are independently H, hydroxyl, mercapto, halogen, cyano, amino, amidine, guanidine, nitro or alkoxy; or R3 and R4 together form a fused carbocycle or heterocycle optionally substituted with hydroxyl, halogen, oxo, thio, amino, amidine, guanidine or alkoxy;
R6 is H or a hydrocarbon chain optionally substituted with a carbocycle or a heterocycle; and
salts, solvates and hydrates thereof;
with the proviso that when Y is phenyl, R2, R4 and R5 are H, R3 is Cl and R1 is OH then X is other than cyclohexyl.
In another aspect of the invention, there is provided pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier.
In another aspect of the invention, there is provided a method of treating a disease or condition mediated by LFA-1 in a mammal comprising administering to said mammal an effective amount of a compound of the invention.
The invention provides novel compounds of formula (I) 
wherein Cy, X, Y, L and R1-6 are as defined herein. Compounds of the invention exhibit reduced plasma protein binding affinity by virtue of a non-aromatic ring at substituent Cy in comparison to those having an aromatic ring at this portion of the molecule.
The term xe2x80x9cnon-aromaticxe2x80x9d refers to carbocycle or heterocycle rings that do not have the properties which define aromaticity. For aromaticity, a ring must be planar, have p-orbitals that are perpendicular to the plane of the ring at each ring atom and satisfy the Huckel rule where the number of pi electrons in the ring is (4n+2) wherein n is an integer (i.e. the number of pi electrons is 2, 6, 10 or 14). Non-aromatic rings provided herein do not satisfy one or all of these criteria for aromaticity.
The term xe2x80x9calkoxyxe2x80x9d as used herein includes saturated, i.e. O-alkyl, and unsaturated, i.e. O-alkenyl and O-alkynyl, group. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy and hexyloxy.
The term xe2x80x9caminoxe2x80x9d refers to a primary (xe2x80x94NH2), secondary (xe2x80x94NHR), tertiary (xe2x80x94N(R)2) or quaternary (xe2x80x94N+(R)4) amine wherein R is a hydrocarbon chain, hydroxy, a carbocycle, a heterocycle or a hydrocarbon chain substituted with a carbocycle or heterocycle.
The term xe2x80x9camino acidxe2x80x9d refers to naturally and non-naturally occurring xcex1-(alpha), xcex2-(beta), D- and L-amino acid residues. Non-natural amino acids include those having side chains other than those occurring in nature.
By xe2x80x9ccarboxylxe2x80x9d is meant herein to be a free acid xe2x80x94COOH as well as esters thereof such as alkyl, aryl and aralkyl esters. Preferred esters are methyl, ethyl, propyl, butyl, i-butyl, s-butyl and t-butyl esters.
The term xe2x80x9ccarbocyclexe2x80x9d refers to a mono-, bi- or tri-cyclic carbon ring or ring system having 4-16 members (including bridged) which is saturated, unsaturated or partially unsaturated including aromatic (aryl) ring systems (unless specified as non-aromatic). Preferred non-aromatic carbocyclic rings include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl. Preferred aromatic carbocyclic rings include phenyl and naphthyl.
The term xe2x80x9cheterocyclexe2x80x9d refers to a mono-, bi- or tri-cyclic ring system having 5-16 members wherein at least one ring atom is a heteroatom (i.e. N, O and S as well as SO, or SO2). The ring system is saturated, unsaturated or partially unsaturated and may be aromatic (unless specified as non-aromatic). Exemplary heterocycles include piperidine, piperazine, pyridine, pyrazine, pyrimidine, pyridazine, morpholine, pyran, pyrole, furan, thiophene (thienyl), imidazole, pyrazole, thiazole, isothiazole, dithiazole, oxazole, isoxazole, dioxazole, thiadiazole, oxadiazole, tetrazole, triazole, thiatriazole, oxatriazole, thiadiazole, oxadiazole, purine and benzofused derivatives thereof.
The term xe2x80x9chydrocarbon chainxe2x80x9d refers to saturated, unsaturated, linear or branched carbon chains i.e. alkyl, alkenyl and alkynyl. Preferred hydrocarbon chains incorporate 1-12 carbon atoms, more preferably 1-6 and most preferably 1-4 carbon atoms i.e. methyl, ethyl, propyl, butyl and allyl.
The phrase xe2x80x9coptionally substituted withxe2x80x9d is understood to mean, unless otherwise stated, that one or more of the specified substituents is covalently attached to the substituted moiety. When more than one, the substituents may be the same or different group.
Cy is a non-aromatic carbocycle or heterocycle optionally substituted with hydroxyl (xe2x80x94OH), mercapto (xe2x80x94SH), thioalkyl, halogen (e.g. F, Cl, Br, I), oxo (xe2x95x90O), thio (xe2x95x90S), amino, aminoalkyl, amidine (xe2x80x94C(NH)xe2x80x94NH2), guanidine (xe2x80x94NH2xe2x80x94C(NH)xe2x80x94NH2), nitro, alkyl or alkoxy. In a particular embodiment, Cy is a 3-5 member ring. In a preferred embodiment, Cy is a 5- or 6-member non-aromatic heterocycle optionally substituted with hydroxyl, mercapto, halogen (preferably F or Cl), oxo (xe2x95x90O), thio (xe2x95x90S), amino, amidine, guanidine, nitro, alkyl or alkoxy. In a more preferred embodiment, Cy is a 5-member non-aromatic heterocycle optionally substituted with hydroxyl, oxo, thio, Cl, C1-4 alkyl (preferably methyl), or C1-4 alkanoyl (preferably acetyl, propanoyl or butanoyl). More preferably the non-aromatic heterocycle comprises one or heteroatoms (N, O or S) and is optionally substituted with hydroxyl, oxo, mercapto, thio, methyl, acetyl, propanoyl or butyl. In particular embodiments the non-aromatic heterocycle comprises at least one nitrogen atom that is optionally substituted with methyl or acetyl. In a particularly preferred embodiment, the non-aromatic heterocycle is selected from the group consisting of piperidine, piperazine, morpholine, tetrahydrofuran, tetrahydrothiophene, oxazolidine, thiazolidine optionally substituted with hydroxy, oxo, mercapto, thio, alkyl or alkanoyl. In a most preferred embodiment Cy is a non-aromatic heterocycle selected from the group consisting of tetrahydrofuran-2-yl, thiazolidin-5-yl, thiazolidin-2-one-5-yl, and thiazolidin-2-thione-5-yl and cyclopropapyrrolidine.
In another preferred embodiment Cy is a 3-6 member carbocycle optionally substituted with hydroxyl, mercapto, halogen, oxo, thio, amino, amidine, guanidine, alkyl, alkoxy or acyl. In a particular embodiment the carbocycle is saturated or partially unsaturated. In particular embodiments Cy is a carbocycle selected from the group consisting of cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl.
X is a C1-5 divalent hydrocarbon linker optionally having one or more carbon atoms replaced with N, O, S, SO or SO2 and optionally being substituted with hydroxyl, mercapto, halogen, amino, aminoalkyl, nitro, oxo or thio. In a preferred embodiment X will have at least one carbon atom. Replacements and substitutions may form an amide moiety (xe2x80x94NRC(O)xe2x80x94 or xe2x80x94C(O)NRxe2x80x94) within the hydrocarbon chain or at either or both ends. Other moieties include sulfonamide (xe2x80x94NRSO2xe2x80x94 or xe2x80x94SO2NR), acyl, ether, thioether and amine. In a particularly preferred embodiment X is the group xe2x80x94CH2xe2x80x94NR6xe2x80x94C(O)xe2x80x94 wherein the carbonyl xe2x80x94C(O)xe2x80x94 portion thereof is adjacent (i.e. covalently bound) to Cy and R6 is alkyl i.e. methyl and more preferably H.
Y is a carbocycle or heterocycle optionally substituted with hydroxyl, mercapto, halogen, oxo, thio, a hydrocarbon, a halo-substituted hydrocarbon, amino, amidine, guanidine, cyano, nitro, alkoxy or acyl. In particular embodiment, Y is aryl or heteroaryl optionally substituted with halogen or hydroxyl. In a particularly preferred embodiment, Y is phenyl, furan-2-yl, thiophene-2-yl, phenyl substituted with a halogen (preferably Cl) or hydroxyl, preferably at the meta position.
L is a divalent hydrocarbon optionally having one or more carbon atoms replaced with N, O, S, SO or SO2 and optionally being substituted with hydroxyl, halogen oxo, or thio; or three carbon atoms of the hydrocarbon are replaced with an amino acid residue. Preferably L is less than 10 atoms in length and more preferably 5 or less and most preferably 5 or 3 atoms in length. In particular embodiments, L is selected from the group consisting of xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NR6xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94NR6xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NR6xe2x80x94CH2xe2x80x94, xe2x80x94CH(OH)xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)2xe2x80x94CH(OH)xe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94C(O)xe2x80x94NR6xe2x80x94CH (R7)xe2x80x94C(O)xe2x80x94NR6xe2x80x94, xe2x80x94NR6xe2x80x94C(O)xe2x80x94CH(R7)xe2x80x94NR6xe2x80x94C(O)xe2x80x94, xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94Oxe2x80x94 and xe2x80x94CH(OH)xe2x80x94CF2xe2x80x94CH2xe2x80x94 wherein each R6 is independently H or alkyl and R7 is an amino acid side chain. Preferred amino acid side chains include non-naturally occurring side chains such as phenyl or naturally occurring side chains. Preferred side chains are those from Phe, Tyr, Ala, Gln and Asn. In a preferred embodiments L is xe2x80x94CHxe2x95x90CHxe2x80x94C(O)xe2x80x94NR6xe2x80x94CH2xe2x80x94 wherein the xe2x80x94CHxe2x95x90CHxe2x80x94 moiety thereof is adjacent (i.e. covalently bound) to Y. In another preferred embodiment, L is xe2x80x94CH2xe2x80x94 NR6xe2x80x94C(O)xe2x80x94 wherein the methylene moiety (xe2x80x94CH2xe2x80x94) thereof is adjacent to Y.
R1 is H, OH, amino, O-carbocycle or alkoxy optionally substituted with amino, a carbocycle or a heterocycle. In a preferred embodiment, R1 is H, phenyl or C1-4 alkoxy optionally substituted with a carbocycle such as phenyl. In a particular embodiment R1 is H. In another particular embodiment R1 is methoxy, ethoxy, propyloxy, butyloxy, isobutyloxy, s-butyloxy, t-butyloxy, phenoxy or benzyloxy. In yet another particular embodiment R1 is NH2. In a particularly preferred embodiment R1 is ethoxy. In another particularly preferred embodiment R1 is isobutyloxy. In another particularly preferred embodiment R1 is alkoxy substituted with amino, for example 2-aminoethoxy, N-morpholinoethoxy, N,N-dialkyaminoethoxy, quaternary ammonium hydroxy alkoxy (e.g. trimethylammoniumhydroxyethoxy).
R2-5 are independently H, hydroxyl, mercapto, halogen, cyano, amino, amidine, guanidine, nitro or alkoxy; or R3 and R4 together form a fused carbocycle or heterocycle optionally substituted with hydroxyl, halogen, oxo, thio, amino, amidine, guanidine or alkoxy. In a particular embodiment R2 and R3 are independently H, F, Cl, Br or I. In another particular embodiment, R4 and R5 are both H. In another particular embodiment, one of R2 and R3 is a halogen while the other is hydrogen or a halogen. In a particularly preferred embodiment, R3 is Cl while R2, R4 and R5 are each H. In another particularly preferred embodiment, R2 and R3 are both Cl while R4 and R5 are both H.
R6 is H or a hydrocarbon chain optionally substituted with a carbocycle or a heterocycle. In a preferred embodiment, R6 is H or alkyl i.e. methyl, ethyl, propyl, butyl, i-butyl, s-butyl or t-butyl. In a particular embodiment R6 is H.
In a preferred embodiment, compounds of the invention have the general formula (Ia)-(If) 
wherein Cy, Y, L and R1-6 are as previously defined. In a particularly preferred embodiment, the carbon atom marked with an asterisk (*) in compounds of formula (Ia)-(If) is chiral. In a particular embodiment, the carbon atom has an R-configuration. In another particular embodiment, the carbon atom has an S-configuration.
Particular compounds of the invention include: 
and salts, solvates, hydrates and esters thereof.
It will be appreciated that compounds of the invention may incorporate chiral centers and therefore exist as geometric and stereoisomers. All such isomers are contemplated and are within the scope of the invention whether in pure isomeric form or in mixtures of such isomers as well as racemates. Stereoisomeric compounds may be separated by established techniques in the art such as chromatography, i.e. chiral HPLC, or crystallization methods.
xe2x80x9cPharmaceutically acceptablexe2x80x9d salts include both acid and base addition salts. Pharmaceutically acceptable acid addition salt refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like, and organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
Compounds of the invention may be prepared according to established organic synthesis techniques from starting materials and reagents that are commercially available or from starting materials that may be prepared from commercially available starting materials. Many standard chemical techniques and procedures are described in March, J., xe2x80x9cAdvanced Organic Chemistryxe2x80x9d McGraw-Hill, New York, 1977; and Collman, J., xe2x80x9cPrinciples and Applications of Organotransition Metal Chemistryxe2x80x9d University Science, Mill Valley, 1987; and Larock, R., xe2x80x9cComprehensive Organic Transformationsxe2x80x9d Verlag, N.Y., 1989. It will be appreciated that depending on the particular substituents present on the compounds, suitable protection and deprotection procedures will be required in addition to those steps described herein. Numerous protecting groups are described in Greene and Wuts, Protective Groups in Organic Chemistry, 2d edition, John Wiley and Sons, 1991, as well as detailed protection and deprotection procedures. For example, suitable amino protecting groups include t-butyloxycarbonyl (Boc), fluorenyl-methyloxycarbonyl (Fmoc), 2-trimethylsilyl-ethyoxy-carbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), allyloxycarbonyl (Alloc), and benzyloxycarbonyl (Cbz). Carboxyl groups can be protected as fluorenyl-methyl groups, or alkyl esters i.e. methyl or ethyl, or alkenyl esters such as allyl. Hydroxyl groups may be protected with trityl, monomethoxytrityl, dimethoxy-trityl, and trimethoxytrityl groups.
Compounds may be prepared according to organic synthetic procedures described in U.S. patent application Ser. No. 09/6446,330 filed on Sep. 14, 2000, the entirety of which is incorporated herein by reference. Generally, compounds may be prepared according to reaction scheme 1.
Referring to scheme 1, a commercially available glycine amino acid residue is protected at the amino (e.g. fmoc) and carboxyl groups (Pr) or else immobilized on a solid support. The amino protecting group is removed with a suitable reagent and is reacted with diphenylketimine and subsequently alkylated at the alpha carbon with (iii) halo-X-Cy to give intermediate (vi). The imine (vi) is converted to the free amine (v) and then coupled with intermediate (vi) to provide the compound of the invention which is optionally deprotected at the carboxyl group to give free acid (vii). The free acid in turn may be esterified or amidated according to the definitions of substituent R1.
In a particular embodiment, compounds of formula (Ib) of the invention may be prepared according to scheme 2.
Referring to scheme 2, starting compound (i), commercially available or synthesized from commercially available reagents, is reacted with N-hydroxymethylphthalamide to give intermediate (ii) which is reacted with hydrazine to yield the free amine (iii). The amine is Boc protected (iv) by reacting with Boc2O and sodiumbicarbonate and then reacted with triflic anhydride to give intermediate (v). The triflate intermediate (v) is then converted to the methyl ester intermediate (vi) by reacting with palladium(II) acetate and 1,3-bi(diphenylphosphino propane (dppp) and subsequently with diisopropyl ethylamine (DIPEA). The Boc group of (vi) is removed with TFA and then reacted with carboxylic acid (vii) to give intermediate (viii). In a preferred embodiment of scheme 2, intermediate (vii) Yxe2x80x94Lxe2x80x94C(O)OH is furylacrylic acid or thienylacrylic acid. The methyl ester of (viii) is removed with LiOH to give the free acid which is reacted with the N-Boc protected diaminopropanoic acid/ester (x) to yield intermediate (xi). The Boc group of (xi) is removed with TFA and then reacted with carboxyl-substituted non-aromatic ring (xii) to give final compound (Ib) of the invention.
In another particular embodiment compounds of formula (Ic) of the invention may be prepared according to scheme 3.
Referring to scheme 3, carboxylate starting reagent (i) is coupled with amine reagent (ii) Yxe2x80x94Lxe2x80x94NHR6 to give intermediate (iii) which is coupled with (iv) to yield compound of the invention (v). In a preferred embodiment of scheme 3, Yxe2x80x94Lxe2x80x94 is benzyl, optionally substituted with hydroxy, halogen, alkyl or alkoxy. More preferably Yxe2x80x94Lxe2x80x94 is 3-hydroxy-benzyl.
In another particular embodiment, compounds of formula (Id) of the invention may be prepared according to scheme 4.
Referring to scheme 4, starting compound (i), prepared according to the procedures described in scheme 2, is converted to the iodo intermediate (ii) and reacted with alkyne (iii) to give intermediate (iv). Alkyne (iii) is prepared by reacting Yxe2x80x94COOH with Brxe2x80x94Cxe2x89xa1CH in THF. Intermediate (iv) is then converted to the alkane (v) by reacting with Rh/Al2O3 in H2 atmosphere and the ester group converted to the free acid by reacting with LiI in pyridine to give (vi). Intermediate (vi) is reacted with amino acid (vii) to give compound of the invention (viii). In a particular embodiment of scheme 4, Y is phenyl optionally substituted with alkyl, hydroxy or halogen. In a particularly preferred embodiment Y is 3-chloro-phenyl or 3-hydroxy-phenyl.
In another particular embodiment, compounds of formula (Ie) of the invention may be prepared according to scheme 5.
Referring to scheme 5, starting compound (i) is reacted with triflic anhydride and 2,6-lutidine to give intermediate (ii) which is converted to methyl ester (iii) by reacting with palladium(II)acetate, 1,3-bi(diphenylphosphino propane (dppp) and subsequently with diisopropyl ethylamine (DIPEA) in DMF and methanol. The ester (iii) is then reacted with CrO3 in acetic acid and anhydride to give aldehyde (iv) which is reacted with Grignard reagent ethynyl-magnesium bromide in THF to give alkyne intermediate (v). Iodo reagent (vi) Yxe2x80x94I is reacted with (v) to give intermediate (vii) which is converted to the alkane (viii) by reacting with Rh/Al2O3 under hydrogen atmosphere. The methyl ester is converted to free acid (ix) with LiI in pyridine which is then coupled to amino acid residue (x) to give compound of the invention (xi). In preferred embodiments of scheme 5, Y is phenyl, optionally substituted with hydroxy, halogen, alkyl or alkoxy. In more preferred embodiments, Y is 3-hydroxy-phenyl or 3-chloro-phenyl.
Compounds of the invention bind to LFA-1 preferentially over Mac-1. Accordingly, in an aspect of the invention, there is provided a method of inhibiting the binding of LFA-1 to ICAMs (cellular adhesion molecules), the method comprising contacting LFA-1 with a compound of formula (I). The method may be carried out in vivo or ex vivo as a solution based or cell based assay wherein the compound of the invention is introduced to LFA-1 in the presence of a putative or known ligand (such as ICAM-1). The compound of the invention may be labeled, for example isotopically radiolabeled, or labeled with a fluorophore such as fluorescein isothiocyanate (FITC), to facilitate detection of ligand binding or reduction thereof to the protease. Thus compounds of the invention are useful for diagnostic and screening assays.
Compounds of the invention are therapeutically and/or prophylactically useful for treating diseases or conditions mediated by LFA-1 activity. Accordingly in an aspect of the invention, there is provided a method of treating a disease or condition mediated by LFA-1 in a mammal, i.e. a human, comprising administering to said mammal an effective amount of a compound of the invention. By xe2x80x9ceffective amountxe2x80x9d is meant an amount of compound which upon administration is capable of reducing the activity of LFA-1; or the amount of compound required to prevent, inhibit or reduce the severity of any symptom associated with an LFA-1 mediated condition or disease upon administration.
Compounds of the invention or compositions thereof are useful in treating conditions or diseases including: psoriasis; responses associated with inflammatory bowel disease (such as Crohn""s disease and ulcerative colitis), dermatitis, meningitis, encephalitis, uveitis, allergic conditions such as eczema and asthma, conditions involving infiltration of T-cells and chronic inflammatory responses, skin hypersensitivity reactions (including poison ivy and poison oak); atherosclerosis, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosis (SLE), diabetes mellitus, multiple sclerosis, Reynaud""s syndrome, autoimmune thyroiditis, experimental autoimmune encephalomyelitis, Sjorgen""s syndrome, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia; diseases involving leukocyte diapedesis; CNS inflammatory disorder, multiple organ injury syndrome secondary to septicaemia or trauma; autoimmune hemolytic anemia; myasthemia gravis; antigen-antibody complex mediated diseases; all types of transplantations, including graft vs. host or host vs. graft disease, HIV and rhinovirus infection, pulmonary fibrosis, alopecia, scleredoma, endometriosus, vitiligo, ischemic reperfusion injury mediated by neutrophils such as acute myocardial infarction, restenosis following PTCA, invasive procedures such as cardiopulmonary bypass surgery, cerebral edema, stroke, traumatic brain injury, hemorragic shock, burns, ischemic kidney disease, multi-organ failure, wound healing and scar formation, atherosclerosis.
The actual amount of compound administered and the route of administration will depend upon the particular disease or condition as well as other factors such as the size, age, sex and ethnic origin of the individual being treated and is determined by routine analysis. In general, intravenous doses will be in the range from about 0.01-1000 mg/kg of patient body weight per day, preferably 0.1 to 20 mg/kg and more preferably 0.3 to 15 mg/kg. Administration may be once or multiple times per day for several days, weeks or years or may be a few times per week for several weeks or years. The amount of compound administered by other routes will be that which provides a similar amount of compound in plasma compared to the intravenous amounts described which will take into consideration the plasma bioavailability of the particular compound administered.
In methods of the invention, the compound may be administered orally (including buccal, sublingual, inhalation), nasally, rectally, vaginally, intravenously (including intra-arterially), intradermally, subcutaneously, intramuscularly and topically. Compounds will be formulated into compositions suitable for administration for example with carriers, diluents, thickeners, adjuvants etc. as are routine in the formulation art. Accordingly, another aspect of the invention provides pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier, excipient or adjuvant and may also include additional active ingredients such as anti-inflammatories e.g. NSAIDs.
Dosage forms include solutions, powders, tablets, capsules, gel capsules, suppositories, topical ointments and creams and aerosols for inhalation. Formulations for non-parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic carrier substances suitable for non-parenteral administration which do not deleteriously react with compounds of the invention can be used. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings flavorings and/or aromatic substances and the like which do not deleteriously react with compounds of the invention. Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. Optionally, the suspension may also contain stabilizers.
Compounds of the invention exhibit high oral bioavailability. Accordingly, in a preferred embodiment, compounds of the invention are administered via oral delivery. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, troches, tablets or SECs (soft elastic capsules or caplets). Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, carrier substances or binders may be desirably added to such formulations. Such formulations may be used to effect delivering the compounds to the alimentary canal for exposure to the mucosa thereof. Accordingly, the formulation can consist of material effective in protecting the compound from pH extremes of the stomach, or in releasing the compound over time, to optimize the delivery thereof to a particular mucosal site. Enteric coatings for acid-resistant tablets, capsules and caplets are known in the art and typically include acetate phthalate, propylene glycol and sorbitan monoleate.
Various methods for producing formulations for alimentary delivery are well known in the art. See, generally Remington""s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990. The formulations of the invention can be converted in a known manner into the customary formulations, such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents. The therapeutically active compound should in each case be present in a concentration of about 0.1% to about 99% by weight of the total mixture, that is to say in amounts which are sufficient to achieve the desired dosage range. The formulations are prepared, for example, by extending the active compounds with solvents and/or excipients, if appropriate using emulsifying agents and/or dispersing agents, and, for example, in the case where water is used as the diluent, organic solvents can be used as auxiliary solvents if appropriate.
Compositions may also be formulated with binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrates (e.g., starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. The preparations may also contain flavoring, coloring and/or sweetening agents as appropriate.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing predetermined amounts of the active ingredients; as powders or granules; as solutions or suspensions in an aqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions or water-in-oil liquid emulsions. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein.